Improved wash compositions

ABSTRACT

Laundry compositions comprising lipases from Psychromonas ingrahamii. Uses of such compositions in methods of laundry, especially at low temperatures.

This application claims priority benefit of EP15183065.0 filed 28 Aug.2015, the contents of which are incorporated herein in their entirety.

The invention relates to compositions comprising certain lipases,especially psychrophilic lipases.

Laundry compositions often contain enzymes to improve cleaningperformance. However, many enzymes are activated only at highertemperatures, which means that large volumes of water must be heated toprovide a wash liquor at an appropriate temperature to activate theenzyme content.

This is proving increasingly unpopular as consumers and laundrycomposition providers focus on sustainability, and as energy pricesrise.

However, despite the obvious appeal of low temperature and cold washes,consumers are often unwilling or reluctant to sacrifice cleaningperformance.

There is a need in the art for laundry compositions having improvedcleaning performance at low temperatures.

SUMMARY

The invention relates to certain lipases and to compositions comprisingsaid lipases. Preferably, the composition also includes a biosurfactant.Biosurfactants, including certain preferred biosurfactants, aredescribed here, as are certain preferred ratios of lipase tobiosurfactant. A preferred biosurfactant is mannosylerythritol lipid(MEL), preferably mannosylerythritol lipid enriched in MEL-B.

Suitably, the lipase is a psychrophilic lipase, for example a coldadapted lipase from Pyschromonas ingrahamii. In other words, suitablythe lipase is a Psychromonas ingrahamii lipase. The inventors haveidentified a putative class 3 lipase from P. ingrahamii termed PinLip.An amino acid sequence alignment is shown in FIG. 1 (aligned withLipex). The Lipex sequence displayed therein may be considered SEQ. ID1,while the PinLip sequence shown therein may be considered SEQ. ID2.

As used herein, the term “psychrophilic” enzyme refers to an enzyme thatis effective at a temperature of 0° C. to 25° C.

Accordingly, the invention further relates to a lipase having an aminoacid sequence as shown in FIG. 1 (labelled Pin-Lip, referred to as SEQ.ID.2), or a sequence identity of at least 70% with the amino acidsequence as shown in FIG. 1 (labelled Pin-Lip, referred to as SEQ.ID.2). Alternatively, this identity may be any of 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, or 100% sequence identity.

Cold active enzymes are desirable as cooler wash liquor temperatures maybe used. This in turn improves sustainability (as heating water forlaundry is a major source of CO₂) and reduces consumer energy bills.This is also useful for laundering articles that may suffer as a resultof high temperature washing, for example by shrinking or fading.

However, a major problem with cold active enzymes is maintaining enzymestability, especially in liquid formulations, during storage, andsubsequently during the wash cycle. Furthermore, cold wash cycles aretypically not suitable for certain stain types, in particular fatstains.

The invention seeks to address at least some of these problems.

In a first aspect, the invention may provide a lipase having a sequenceidentity of at least 70% with SEQ. ID. 2.

In a further aspect, the invention may provide a laundry compositioncomprising such a lipase.

In a further aspect, the invention may provide a laundry compositioncomprising a lipase from Pyschromonas ingrahamii. The lipase may bereferred to as a psychrophilic lipase and/or a cold adapted lipase.

The lipase may be a putative class 3 lipase. In other words, the lipasemay be a putative triglyceride lipase. Putative in this case meansidentified as such using tests known in the art.

The inventors have shown that the inclusion of a biosurfactant improvescleaning when compared to the psychrophilic lipase alone. The inventorshave also shown that the combination of psychrophilic lipase andbiosurfactant is, in at least some cases, superior to a combination ofthe benchmark mesophilic lipase Lipex and the same biosurfactants, inparticular at lower temperatures. Accordingly, preferably thecomposition also includes a biosurfactant.

The psychrophilic lipase a Psychromonas ingrahamii lipase. The lipasemay be wild-type or mutant. The lipase may have an amino acid sequenceas shown in FIG. 1 (labelled Pin-Lip), or a sequence identity of atleast 70% with the amino acid sequence as shown in FIG. 1.Alternatively, this identity may be any of 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, or 100% sequence identity. For example, it may be atleast 75%, at least 80%, at least 85%, at least 90%, or even at least95%.

Preferably, the composition is a liquid. Liquid compositions arepreferred by many consumers, and concentrated liquid products improvesustainability owing to decreased packaging and a smaller transportationfootprint.

Suitably, the composition comprises a biosurfactant which is aglycolipid (in other words, the biosurfactant comprises a carbohydrate).Suitable biosurfactants are as described herein and include rhamnolipid,sophorolipid, trehalolipid (trehalose lipids), and a mannosylerythritollipid (MEL), and combinations thereof.

It will be appreciated that each of these terms refers to a known classof compounds. The glycolipid may be of a single structure, for example,MEL-B, or it may be a mixture of structures within the class.

In some cases, the biosurfactant is a rhamnolipid. The rhamnolipid maycomprise at least 50 wt. % monorhamnoplipid, optionally at least 60 wt.%, 70 wt. %, 80 wt. %, 90 wt. %, 95 wt. %, 98 wt. %, even up to 100 wt.%. The rhamnolipid may comprise at least 50 wt. % di-rhamnolipid,optionally at least 60 wt. %, 70 wt. %, 80 wt. %, 90 wt. %, 95 wt. %, 98wt. %, even up to 100 wt. %. Preferably, the rhamnolipid is enriched inmonorhamnolipid. For example, the rhamnolipid may comprise at least 50wt. % monorhamnolipid, for example at least 80 wt. % monorhamnolipid.The biosurfactant may be exclusively monorhamnolipid.

In some cases, the biosurfactant is a mannosylerythritol lipid.Preferably, the mannosylerythritol lipid in enriched inmannosylerythritol lipid B (MEL-B). The MEL may comprises at least 50wt. % MEL-B, optionally at least 60 wt. %, 70 wt. %, 80 wt. %, 90 wt. %,95 wt. %, 98 wt. %, even up to 100 wt. %. The biosurfactant may beexclusively MEL-B.

The inventors have observed that compositions comprising a psychrophiliclipase as described herein and a biosurfactant provide enhanced cleaningat lower total surfactancy when compared to the benchmark enzyme Lipexand a biosurfactant. Laundry compositions comprising a psychrophiliclipase as described herein therefore offer potential for improved stainremoval at low temperature.

In some cases, the total surfactant content of the composition is 30 wt.% or less, for example, 25% or less, 20% or less, 18% or less, 15% orless, 12% or less or even lower.

It will be appreciated that surfactants other than biosurfactants may bepresent in the compositions. In other words, the composition maycomprise glycolipid surfactants and/or other surfactants. In some cases,the total surfactant content is glycolipid surfactant content. In othercases, there is a mixture of glycolipid surfactant and other surfactant.In other cases, no biosurfactant is present. Other surfactants are knownin the art and include linear alkylbenzene sulfonic acid (LAS), sodiumlaureth sulfate (SLES) and non-ionic surfactants.

In some cases, the ratio of biosurfactant to non-biosurfactant may befrom 1:9 to 1:1, for example, from 1:9 to 1:2, from 1:9 to 1:3, from 1:9to 1:4.

In other words, the biosurfactant content may be from 1 to 100 wt. % ofthe total surfactant content of the composition. In some cases, thebiosurfactant content is from 1 to 50 wt. % of the total surfactantcontent of the composition, for example, from 10 to 50 wt. % of thetotal surfactant content of the composition. In some cases it is 10 wt.%, 20 wt. %, 30 wt. %, 40 wt. %, or 50 wt. %.

The non-biosurfactants (other surfactants) may include, withoutlimitation LAS (linear alkylbenzene sulfonate), SLES (sodium laurylether sulfate), and NI (non-ionic surfactants). For example, in somecases the ratio of LAS to SLES to NI is 2:1:3.

Suitably, the ratio of lipase to biosurfactant is from 1:10 to 1:200,for example, from 1:10 to 1:150, from 1:10 to 1:100, from 1:15 to 1:80,from 1:20 to 1:60, from 1:30 to 1:50. In some cases, it is around 1:40.

The compositions of the invention permit articles (such as clothes,curtains, household linen, and towels) to be laundered at lowertemperatures.

The invention may further provide a composition comprising a lipase anda mannosylerythritol lipid. Preferably, the mannosylerythritol lipid inenriched in mannosylerythritol lipid B (MEL-B). The MEL may comprises atleast 50 wt. % MEL-B, optionally at least 60 wt. %, 70 wt. %, 80 wt. %,90 wt. %, 95 wt. %, 98 wt. %, even up to 100 wt. %. The biosurfactantmay be exclusively MEL-B. Except where expressly provided otherwise, thepreferences described with respect to the first aspect also apply here.

In a further aspect, the present invention relates to a method oflaundering articles, the method comprising washing articles in anaqueous wash liquor containing a composition according to the firstaspect. For example, the temperature of the water is room temperature(also referred to as ambient temperature).

Advantageously, cooler washing steps may be used owing to the desirablestain removal and cleaning at low temperatures facilitated combinationof the invention. For example, even for fat staining, the washing steptemperature may be 40° C. or less, 35° C. or less, 30° C. or less, 25°C. or less. In some preferred embodiments, no heating is used (unheatedwater is used): the wash liquor temperature is the temperature of thecold fill into the machine or from the faucet into a bowl or basin.Naturally, this will vary with supply and geographical variation, butmay be as low as 10° C., or even lower. For example, in northern USstates the water supply may be as low as 7° C. or even 5° C. in winter.This may be referred to as an ambient wash.

Not heating the water reduces energy consumption, reducing energy billsand making laundry more environmentally friendly.

Accordingly, in a further aspect, the invention provides a method oflaundering articles, the method comprising washing articles in anaqueous wash liquor containing a composition according to as describedherein, wherein the temperature of the wash liquor is 25° C. or less,20° C. or less, 15° C. or less, 10° C. or less, or even 5° C. or less.

The inventors have found that for at least some psychrophilic enzymessuch as PinLip, the activity for certain short/medium length estersimproves at the upper temperature range of such an ambient wash.Accordingly, in some cases, the temperature of the wash liquor is 15-25°C.

However, the inventors have observed that across a wide variety oftemperature ranges, the lipase is active for a range of ester chainlengths.

The inventors have found that low concentrations of psychrophilic lipasemay be used. For example, the concentration of psychrophilic lipase inthe wash liquor may be 2.5 to 20 mg/L.

This further improves sustainability and economy.

Suitably, the concentration of biosurfactant in the wash liquor is 0.001to 1 wt %, preferably 0.005 to 0.5 wt %, 0.01 to 0.5 wt %, 0.01 to 0.2wt %.

DRAWINGS

FIG. 1 shows the sequence alignment of a putative class 3 lipase from P.ingrahammii.

FIG. 2 shows a 10% SDS-PAGE gel of purified PinLip.

FIG. 3 shows a schematic reaction of pNp-ester hydrolysis performed by alipase.

FIG. 4 shows linearisation of pNp calibration curve.

FIG. 5 shows PinLip activity towards different pNp-esters at: 5a: 4° C.;5b:15° C.; 5c: 25° C.

FIG. 6 shows a comparison of PinLip and Lipex in combination with aformulation (Blackbull), and the control of the formulation alone.

FIG. 7 shows SRI values at a variety of enzyme concentrations for bothPinLip (a) and Lipex (b).

FIG. 8 shows the SRI values for PinLip in combination with variousbiosurfactants at differing concentrations of biosurfactant.

FIG. 9 shows SRI values for varying ratios of biosurfactant tosurfactant.

DESCRIPTION Abbreviations

Suc-Ala-Ala-Phe-7-amido-4-methylcoumarin—N-SUCCINYL-L-ALANYL-L-ALANYL-L-PHENYLALANINE-4-METHYL-COUMARYL-7-AMIDE

CAPS—N-cyclohexyl-3-aminopropanesulfonic acid

Tris—tris(hydroxymethyl)aminomethane

CIE—‘Commission Internationale de l'Eclairage’

SRI—Stain Removal Index

Definitions

As used herein the term “effective” means that the enzyme has theability to achieve stain removal or catalytic capability (in the giventemperature zone).

As used herein the term “treatment” in the context of enzymatic fabrictreatment composition preferably means cleaning and more preferablystain removal.

Preferably stain removal is measured in terms of Remission units or aRemission index. Effective stain removal is preferably represented byremission equal to or greater than 2 Remission units.

Enzymes

As used herein the term “enzyme” includes enzyme variants (produced, forexample, by recombinant techniques). Examples of such enzyme variantsare disclosed, e.g., in EP 0251446 (Genencor), WO 91/00345 (NovoNordisk), EP 0525610 (Solvay) and WO 94/02618 (Gist-Brocades NV), eachof which is incorporated by reference in its entirety.

Enzymes may be from bacterial or fungal origin. Chemically modified orprotein engineered mutants are included.

Percentage Sequence Identity

Percentage (%) sequence identity is defined as the percentage of aminoacid residues in a candidate sequence that are identical with residuesin the given listed sequence (referred to by the SEQ ID No.) afteraligning the sequences and introducing gaps if necessary, to achieve themaximum sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Sequence identity ispreferably calculated over the entire length of the respectivesequences.

Where the aligned sequences are of different length, sequence identityof the shorter comparison sequence may be determined over the entirelength of the longer given sequence or, where the comparison sequence islonger than the given sequence, sequence identity of the comparisonsequence may be determined over the entire length of the shorter givensequence.

For example, where a given sequence comprises 100 amino acids and thecandidate sequence comprises 10 amino acids, the candidate sequence canonly have a maximum identity of 10% to the entire length of the givensequence. This is further illustrated in the following example:

(A) Given seq: XXXXXXXXXXXXXXX (15 amino acids) Comparison seq:XXXXXYYYYYYY (12 amino acids)

The given sequence may, for example, be that encoding Pin Lip as shownin FIG. 1.

% sequence identity=the number of identically matching amino acidresidues after alignment divided by the total number of amino acidresidues in the longer given sequence, i.e. (5 divided by 15)×100=33.3%

Where the comparison sequence is longer than the given sequence,sequence identity may be determined over the entire length of the givensequence. For example:

(B) Given seq: XXXXXXXXXX (10 amino acids) Comparison seq:XXXXXYYYYYYZZYZZZZZZ (20 amino acids)

Again, the given sequence may, for example, be that encoding Pin Lip asshown in FIG. 1.

% sequence identity=number of identical amino acids after alignmentdivided by total number of amino acid residues in the given sequence,i.e. (5 divided by 10)×100=50%.

Alignment for purposes of determining percent amino acid sequenceidentity can be achieved in various ways known to a person of skill inthe art, for instance, using publicly available computer software suchas ClustalW 1.82. T-coffee or Megalign (DNASTAR) software. When usingsuch software, the default parameters, e.g. for gap penalty andextension penalty, are preferably used. The default parameters ofClustalW 1.82 are: Protein Gap Open Penalty=10.0, Protein Gap ExtensionPenalty=0.2, Protein matrix=Gonnet, Protein/DNA ENDGAP=−1, Protein/DNAGAPDIST=4.

Identity of nucleic acid sequences may be determined in a similar mannerinvolving aligning the sequences and introducing gaps if necessary, toachieve the maximum sequence identity, and calculating sequence identityover the entire length of the respective sequences. Where the alignedsequences are of different length, sequence identity may be determinedas described above and illustrated in examples (A) and (B).

Psychrophilic Enzymes

As used herein the term “psychrophilic enzyme” means enzymes that areeffective at a temperature of 0° C.-25° C.

As used herein the term “effective” means that the enzyme has theability to achieve stain removal or catalytic capability (in the giventemperature zone). Preferably stain removal is measured in terms ofRemission units or a Remission index. Effective stain removal ispreferably represented by remission equal to or greater than 2 Remissionunits.

In some cases, the psychrophilic enzyme is effective at a temperature of0° C.-20° C., for example at a temperature of 0° C.-15° C. In somecases, the psychrophilic enzyme is effective at a temperature of 0°C.-10° C.

Preferably the psychrophilic enzyme comprises e.g. a lipase and/or aphospholipase.

Lipases are highly advantageous psychrophilic enzymes because fats andoil based stains are more difficult to remove at psychrophilictemperatures. Phospholipases are advantageous psychrophilic enzymes formuch the same reason.

Psychrophilic lipases include lipases from Acinetobacter sp. Strain No.6 (Suzuki et al. (2001) J. Biosci. Bioeng. 92: 144-148); Acinetobactersp. Strain No. 016 (Brueuil and Kushner, (1975) Can. J. Microbiol.21:423-433); Achromobacter lipolyticum (Khan et al., (1967), Biochem.Biophys. Acta. 132:68-77 1967), Aeromonas sp. Strain No. LPB 4 (Lee etal. (2003), J. Microbiol. 41:22-27), Aeromonas hydrophila (Pemberton etal. (1997) FEMS Microbiol. Lett. 152:1-10); Bacillus sphaericus MTCC7526 (Joseph. PhD Thesis (2006) Allahabad Agricultural Institute,Allahabad, Ind.); Microbacterium phyllosphaerae MTCC 7530, Moraxella sp.(Feller et al. (1990) FEMS Microbiol. Lett. 66:239-244; Moraxella sp.TA144 (Feller et al. (1991) Gene. 102:111-115); Photobacteriumlipolyticum M37 (Ryu et al. (2006) Appl. Microbiol. Biotechnol. 70:321-326); Pseudoalteromonas sp. Wp27 (Zeng et al. (2004) J. Microbiol.Biotechnol. 14: 952-958); Pseudoalteromonas sp. (Giudice et al. (2006)J. Applied Microbiology 101:1039-1048), Psychrobacter sp. and Vibriosp.; Psychrobacter sp. Wp37 (Zeng et al. (2004) J. Microbiol.Biotechnol. 14: 952-958); Psychrobacter okhotskensis sp. (Yumoto et al.(2003) Int. J. Syst. Evol. Microbiol. 53: 1985-1989); Psychrobacter sp.Ant300 (Kulakovaa et al. (2004) Biochemica. Biophysica. Acta.1696:59-65); Psychrobacter immobilis strain B 10 (Arpigny et al. (1997)J. Mol. Catal. B Enzy. 3: 29-35), Psychromonas ingrahamii (Gosink et al.(1993) FEMS Microbiol Ecol 102, 85-90; Serratia marcescens (Abdou,(2003) J. Dairy Sci. 86:127-132), Staphylococcus aureus (Alford andPierce, (1961) J. Food Sci. 26:518-524), and Staphylococcus epidermidis(Joseph et al. (2006) J. Gen. Appl. Microbiol. 52: 315-320). Eachdocument is incorporated by reference in its entirety for all purposes,but in particular the disclosure of psychrophilic enzyme identity,structure, reactivity and methods of obtaining said enzymes.

Preferably, the psychrophilic lipase is a class 3 lipase fromPsychromonas ingrahamii (known as PinLip).

Mesophilic Lipases

Exemplary mesophilic lipases include lipases from Humicola (synonymThermomyces), e.g. from H. lanuginosa (T. lanuginosus) or from H.insolens, a Pseudomonas lipase, e.g. from P. alcaligenes or P.pseudoalcaligenes, P. cepacia, P. stutzeri, P. fluorescens, Pseudomonassp. strain SD 705 (WO 95/06720 and WO 96/27002), P. wisconsinensis, aBacillus lipase, e.g. from B. subtilis (Dartois et al. (1993),Biochemica et Biophysica Acta, 1131, 253-360), B. stearothermophilus (JP64/744992) or B. pumilus (WO 91/16422). Each document is incorporated byreference in its entirety for all purposes, but in particular thedisclosure of enzyme identity, structure, reactivity and methods ofobtaining said enzymes.

Commercially available mesophilic lipase enzymes include Lipolase™ andLipolase Ultra™, Lipex™ (Novozymes A/S).

Exemplary mesophilic phospholipases (EC 3.1.1.4 and/or EC 3.1.1.32)include enzymes which hydrolyse phospholipids. Phospholipases A₁ and A₂which hydrolyze one fatty acyl group (in the sn-1 and sn-2 position,respectively) to form lysophospholipid; and lysophospholipase (orphospholipase B) which can hydrolyze the remaining fatty acyl group inlysophospholipid are included as are Phospholipase C and phospholipase D(phosphodiesterases) which release diacyl glycerol or phosphatidic acidrespectively.

The term “phospholipase A” used herein in connection with an enzyme ofthe invention is intended to cover an enzyme with Phospholipase A₁and/or Phospholipase A₂ activity. The phospholipase activity may beprovided by enzymes having other activities as well, such as, e.g., alipase with phospholipase activity.

The mesophilic phospholipase may be of any origin, e.g., of animalorigin (such as, e.g., mammalian), e.g. from pancreas (e.g., bovine orporcine pancreas), or snake venom or bee venom. Preferably thephospholipase may be of microbial origin, e.g., from filamentous fungi,yeast or bacteria, such as the genus or species Aspergillus, e.g., A.niger; Dictyostelium, e.g., D. discoideum; Mucor, e.g. M. javanicus, M.mucedo, M. subtilissimus; Neurospora, e.g. N. crassa; Rhizomucor, e.g.,R. pusillus; Rhizopus, e.g. R. arrhizus, R. japonicus, R. stolonifer;Sclerotinia, e.g., S. libertiana; Trichophyton, e.g. T. rubrum;Whetzelinia, e.g., W. sclerotiorum; Bacillus, e.g., B. megaterium, B.subtilis; Citrobacter, e.g., C. freundii; Enterobacter, e.g., E.aerogenes, E. cloacae Edwardsiella, E. tarda; Erwinia, e.g., E.herbicola; Escherichia, e.g., E. coli; Klebsiella, e.g., K. pneumoniae;Proteus, e.g., P. vulgaris; Providencia, e.g., P. stuartii; Salmonella,e.g. S. typhimurium; Serratia, e.g., S. liquefasciens, S. marcescens;Shigella, e.g., S. flexneri; Streptomyces, e.g., S. violeceoruber;Yersinia, e.g., Y. enterocolitica. Thus, the phospholipase may befungal, e.g., from the class Pyrenomycetes, such as the genus Fusarium,such as a strain of F. culmorum, F. heterosporum, F. solani, or a strainof F. oxysporum. The phospholipase may also be from a filamentous fungusstrain within the genus Aspergillus, such as a strain of Aspergillusawamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nigeror Aspergillus oryzae.

Preferred mesophilic phospholipases are derived from a strain ofHumicola, especially Humicola lanuginosa or variant; and from strains ofFusarium, especially Fusarium oxysporum. The phospholipase may bederived from Fusarium oxysporum DSM 2672.

Preferably mesophilic phospholipases comprise a phospholipase A₁ (EC.3.1.1.32). or a phospholipase A₂ (EC.3.1.1.4.).

Examples of commercial mesophilic phospholipases include LECITASE™ andLECITASE™ ULTRA, YIELSMAX, or LIPOPAN F (available from Novozymes A/S,Denmark).

Other Enzymes

Advantageously, alternatively, or additionally, the psychrophilic enzymecan comprise an esterase (ester hydrolase) such as a carboxylic esterhydrolase. For example, the enzyme can include a glycosyl hydrolase(glycosylase) for example a cellulase, an amylase (includingalpha-amylases), a xylanase, etc.

Psychrophilic esterases preferably include esterases EstAT1 and EstAT11described by Jeon et al. Mar Biotechnol (2009) 11:307-316, which isincorporated by reference in its entirety for all purposes, but inparticular the disclosure of enzyme identity, structure, reactivity andmethods of obtaining said enzymes.

Psychrophilic glycosyl hydrolases preferably include glycosidases suchas amylases, e.g. α-amylases from Pseudoalteromonas haloplanktis strainTAC 125 and from Alteromonas haloplanktis A23 (Feller et al (1998)Journal Biological Chemistry Vol 273, No. 20 pp 12109-12115) and fromNocardiopsis sp. 7326; cellulases and xylanase from e.g. Clostridium sp.PXYL1 (G. Akila, T. S. Chandra (2003) FEMS Microbiol. Letters 219,63-67). Psychrophilic xylanases include E. coli phagemid (Lee et al.2006b). Each document is incorporated by reference in its entirety forall purposes, but in particular the disclosure of enzyme identity,structure, reactivity and methods of obtaining said enzymes.

Exemplary psychrophilic proteases include those derived fromFlavobacterium balustinum P104 (isolated from the internal organs ofsalmon and has been deposited in National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology as thedeposit number of FERM BP-5006 on Feb. 17, 1995 and described inWO/1996/025489) and from Arthrobacter globiformis S155 (Poitier et al,(1995) J. Gen. Microbiol. 133:2797-2806). Each document is incorporatedby reference in its entirety for all purposes, but in particular thedisclosure of enzyme identity, structure, reactivity and methods ofobtaining said enzymes.

Psychrophilic lyases preferably include pectate lyases e.g. fromPseudoalteromonas haloplanktis strain ANT/505 (Truong et al (2001)Extremophiles 5: 35-44).

Preferably, the one or more mesophilic enzymes comprise proteases and/orglycosidases and/or pectate lyases.

Mesophilic proteases include serine protease or a metallo protease,preferably an alkaline microbial protease or a trypsin-like protease.Alkaline proteases include subtilisins, especially those derived fromBacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309,subtilisin 147 and subtilisin 168. The protease may be trypsin-like(i.e. capable of cleaving peptide bonds at the C-terminal side of lysineor arginine). Such proteases may be of porcine or bovine origin.Fusarium derived trypsin proteases are also included.

Commercially available protease enzymes include Alcalase™, Savinase™,Primase™, Duralase™, Dyrazym™, Esperase™, Everlase™, Polarzyme™, andKannase™, (Novozymes A/S), Maxatase™, Maxacal™, Maxapem™, Properase™,Purafect™, Purafect OxP™, FN2™, and FN3™ (Genencor International Inc.).

Exemplary mesophilic cutinases (EC 3.1.1.74) are derived from a strainof Aspergillus, in particular Aspergillus oryzae, a strain ofAlternaria, in particular Alternaria brassiciola, a strain of Fusarium,in particular Fusarium solani, Fusarium solani pisi, Fusarium roseumculmorum, or Fusarium roseum sambucium, a strain of Helminthosporum, inparticular Helminthosporum sativum, a strain of Humicola, in particularHumicola insolens, a strain of Pseudomonas, in particular Pseudomonasmendocina, or Pseudomonas putida, a strain of Rhizoctonia, in particularRhizoctonia solani, a strain of Streptomyces, in particular Streptomycesscabies, or a strain of Ulocladium, in particular Ulocladiumconsortiale. Most preferably cutinase is derived from a strain ofHumicola insolens, in particular the strain Humicola insolens DSM 1800.

Commercial cutinases include NOVOZYM™ 51032 (available from NovozymesA/S, Denmark).

Exemplary mesophilic amylases (alpha and/or beta) are included forexample, alpha-amylases obtained from Bacillus, e.g. from strains of B.licheniformis NCIB8059, ATCC6634, ATCC6598, ATCC11945, ATCC 8480,ATCC9945a, or the Bacillus sp. strains DSM 12649 (AA560 alpha-amylase)or Bacillus sp. DSM 12648 (AA349 alpha-amylase).

Commercially available mesophilic amylases are Duramyl™, Termamyl™,Termamyl Ultra™, Natalase™, Stainzyme™, Fungamyl™ and BAN™ (NovozymesANS), Rapidase™ and Purastar™ (from Genencor International Inc.).

Exemplary mesophilic cellulases include cellulases from the generaBacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g.the fungal cellulases produced from Humicola insolens, Thielaviaterrestris, Myceliophthora thermophila, and Fusarium oxysporum.

Especially preferred mesophilic cellulases are the alkaline or neutralcellulases having color care benefits. Commercially available cellulasesinclude Celluzyme™, Carezyme™, Endolase™, Renozyme™ (Novozymes ANS),Clazinase™ and Puradax HA™ (Genencor International Inc.), andKAC-500(B)™ (Kao Corporation).

Exemplary mesophilic pectate lyases include pectate lyases that arederived/cloned from bacterial genera such as Erwinia, Pseudomonas,Klebsiella and Xanthomonas, as well as from Bacillus subtilis (Nasser etal. (1993) FEBS Letts. 335:319-326) and Bacillus sp. YA-14 (Kim et al.(1994) Biosci. Biotech. Biochem. 58:947-949); Bacillus pumilus (Dave andVaughn (1971) J. Bacteriol. 108:166-174), B. polymyxa (Nagel and Vaughn(1961) Arch. Biochem. Biophys. 93:344-352), B. stearothermophilus(Karbassi and Vaughn (1980) Can. J. Microbiol. 26:377-384), Bacillus sp.(Hasegawa and Nagel (1966) J. Food Sci. 31:838-845) and Bacillus sp. RK9(Kelly and Fogarty (1978) Can. J. Microbiol. 24:1164-1172. Each documentis incorporated by reference in its entirety for all purposes, but inparticular the disclosure of enzyme identity, structure, reactivity andmethods of obtaining said enzymes. Divalent cation-independent and/orthermostable pectate lyases may be used.

Examples of commercially available alkaline mesophilic pectate lyasesinclude BIOPREP™ and SCOURZYME™ L from Novozymes ANS, Denmark.

Exemplary mesophilic mannanases (EC 3.2.1.78) include derived from astrain of the filamentous fungus genus Aspergillus, preferablyAspergillus niger or Aspergillus aculeatus or Trichoderma reseei or fromthe Bacillus microorganism FERM P-8856 which produces beta-mannanase andbeta-mannosidase or from alkalophilic Bacillus sp. AM-001 or fromBacillus amyloliquefaciens. The mannanase may comprise alkaline family 5and 26 mannanases derived from Bacillus agaradhaerens, Bacilluslicheniformis, Bacillus halodurans, Bacillus clausii, Bacillus sp., andHumicola insolens.

Examples of commercially available mannanases include Mannaway™available from Novozymes A/S Denmark.

Exemplary mesophilic peroxidases/oxidases include peroxidases fromCoprinus, e.g. from C. cinereus, and variants thereof. Commerciallyavailable peroxidases include Guardzyme™ and Novozym™ 51004 (NovozymesA/S).

Thermophilic Enzymes

Thermophilic proteases include proteases derived from ThermophilicBacillus strain HS08 (African Journal of Biotechnology Vol. 5 (24), pp.2433-2438, 18 Dec. 2006) and B. Stearothermophilius 1503; Thermoscaldophilus GK24; T. Aquaticus T351; T. aquaticus YT1 Aq.I and Aq. II.

Thermophilic lipases include those derived from Bacillusthermocatenulatus BTL1 and preferably BTL2 (Schimdt-Dannert et al,Biochim. Biophys. Acta (1994) 1214, pp. 43-5 and Biochim. Biophys. Acta(1996) 1301, pp. 105-114). Thermophilic glycosyl hydrolases includealpha-amylases from B. stearothermophilus Donk, strain BS-1 (JournalBiochemistry, Vol 67, 1:65-75) and from Bacillus sp. ANT-6 (ProcessBiochemistry (May 2003) Vol 38, 10:1397-1403). Thermophilic lyasesinclude the pectate lyases from Thermoanaerobacter italicus sp. nov.strain Ab9 (Kozianowski et al., (1997) Extremophiles Vol 1, 4:171-182).Each document is incorporated by reference in its entirety for allpurposes, but in particular the disclosure of enzyme identity,structure, reactivity and methods of obtaining said enzymes.

Once each suitable enzyme is chosen according to the invention, it isrelatively easy for the skilled man to isolate a suitable micro-organismcapable of producing the enzyme and to carry out optimization proceduresknown in the art for making enzymes which have the requiredstability/performance in e.g. powder or liquid compositions and/or incertain washing conditions etc.

Surfactant

The composition preferably comprises a surfactant.

Preferably the composition comprises a detersive surfactant. By adetersive surfactant it is meant that the surfactant, or at least onesurfactant of any surfactant mixture, provides a detersive, i.e.cleaning effect to textile fabrics treated as part of a launderingprocess. Other surfactants, which may or may not be detersivesurfactants, can be used as part of the composition.

The surfactant may be a synthetic surfactant or a biosurfactant which ismicrobially synthesized e.g. from bacteria, fungi or other microbe.

The biosurfactant preferably comprises a microbially-derivedbiosurfactant. Preferably it comprises a glycolipid biosurfactant whichmay be a rhamnolipid or sophorolipid or trehalolipid or amannosylerythritol lipid (MEL). Alternatively, the biosurfactant mayadvantageously comprise a cellobiose, peptide based biosurfactants,lipoproteins and lipopeptides e.g. surfactin, fatty acids e.g.corynomucolic acids (preferably with hydrocarbon chain C12-C14),phospholipids e.g. Phosphatidylethanolamine produced by Rhodococcuserythropolis grown on n-alkane resulted in the lowering of interfacialtension between water and hexadecane to less than 1 mN m−1 and CMC of 30mg L−1 (Kretschner et al., 1982) and Spiculisporic acid; polymericbiosurfactants including emulsan, liposan, mannoprotein andpolysaccharide-protein complexes. Preferably the biosurfactant comprisesa rhamnolipid.

The surfactant is present by weight in the laundry detergentcompositions at a level of from 3 to 85% by weight, preferably from 3 to60% by weight, more preferably from 3 to 40% by weight, most preferablyfrom 3 to 35% by weight. Additional surfactants can also be incorporatedin the laundry compositions of the invention; these may be detersive ornon-detersive surfactants.

Preferably the surfactant comprises anionic surfactant, nonionicsurfactant or a mixture of the two. More preferably the surfactantmixture comprises anionic and nonionic surfactants. Cationic surfactantmay optionally be present as part of the surfactant.

If present, anionic surfactant is present at a level of from 0.1 to 95%by weight, preferably from 1 to 50% by weight, more preferably from 1.5to 25% by weight based on total weight of surfactants present. Nonionicsurfactant, if present, is incorporated at a level of from 0.1 to 95% byweight, preferably from 1 to 50% by weight, more preferably from 1.5 to25% by weight based on total weight of surfactants present. If asurfactant mixture is used that incorporates both anionic and nonionicsurfactants, then preferably the ratio of anionic surfactant to nonionicsurfactant is from 10:1 to 1:10.

Nonionic Surfactant

For the purposes of this disclosure, ‘nonionic surfactant’ shall bedefined as amphiphilic molecules with a molecular weight of less thanabout 10,000, unless otherwise noted, which are substantially free ofany functional groups that exhibit a net charge at the normal wash pH of6-11.

Any type of nonionic surfactant may be used. Highly preferred are fattyacid alkoxylates, especially ethoxylates, having an alkyl chain of fromC₈-C₃₅, preferably C₈-C₃₀, more preferably C₁₀-C₂₄, especially C₁₀-C₁₈carbon atoms, and having preferably 3 to 25, more preferred 5 to 15ethylene oxide groups, for example, Neodols from Shell (The Hague, TheNetherlands); ethylene oxide/propylene oxide block polymers which mayhave molecular weight from 1,000 to 30,000, for example, Pluronic(trademark) from BASF (Ludwigshafen, Germany); and alkylphenolethoxylates, for example Triton X-100, available from Dow Chemical(Midland, Mich., USA).

Other nonionic surfactants considered within the scope of this inventioninclude condensates of alkanolamines with fatty acids, such as cocamideDEA, polyol-fatty acid esters, such as the Span series available fromUniqema (Gouda, The Netherlands), ethoxylated polyol-fatty acid esters,such as the Tween series available from Uniqema (Gouda, TheNetherlands), alkylpolyglucosides, such as the APG line available fromCognis (Düsseldorf, Germany) and n-alkylpyrrolidones, such as theSurfadone series of products marketed by ISP (Wayne, N.J., USA).

Anionic Surfactant

‘Anionic surfactants’ are defined herein as amphiphilic moleculescomprising one or more functional groups that exhibit a net anioniccharge when in aqueous solution at the normal wash pH of between 6 and11.

Preferred anionic surfactants are the alkali metal salts of organicsulphur reaction products having in their molecular structure an alkylradical containing from about 6 to 24 carbon atoms and a radicalselected from the group consisting of sulphonic and sulphuric acid esterradicals.

Although any anionic surfactant hereinafter described can be used, suchas alkyl ether sulphates, soaps, fatty acid ester sulphonates, alkylbenzene sulphonates, sulphosuccinate esters, primary alkyl sulphates,olefin sulphonates, paraffin sulphonates and organic phosphate;preferred anionic surfactants are the alkali and alkaline earth metalsalts of fatty acid carboxylates, fatty alcohol sulphates, preferablyprimary alkyl sulfates, more preferably they are ethoxylated, forexample alkyl ether sulfates; and alkylbenzene sulfonates or mixturesthereof.

Cationic, Amphoteric Surfactants and/or Zwitterionic Surfactants

Also cationic, amphoteric surfactants and/or zwitterionic surfactantsmay be present in the compositions according to the invention.

Preferred cationic surfactants are quaternary ammonium salts of thegeneral formula R₁R₂R₃R₄N⁺ X⁻, for example where R₁ is a C₁₂-C₁₄ alkylgroup, R₂ and R₃ are methyl groups, R₄ is a 2-hydroxyethyl group, and X⁻is a chloride ion. This material is available commercially as Praepagen(Trade Mark) HY from Clariant GmbH, in the form of a 40% by weightaqueous solution.

In a preferred embodiment the composition according to the inventioncomprises an amphoteric or zwitterionic surfactant. Amphotericsurfactants are molecules that contain both acidic and basic groups andwill exist as zwitterions at the normal wash pH of between 6 and 11.Preferably an amphoteric or zwitterionic surfactant is present at alevel of from 0.1 to 20% by weight, more preferably from 0.25 to 15% byweight, even more preferably from 0.5 to 10% by weight.

Suitable zwitterionic surfactants are exemplified as those which can bebroadly described as derivatives of aliphatic quaternary ammonium,sulfonium and phosphonium compounds with one long chain group havingabout 8 to about 18 carbon atoms and at least one water solubilizingradical selected from the group consisting of sulfate, sulfonate,carboxylate, phosphate or phosphonate. A general formula for thesecompounds is:

R₁(R₂)_(x)Y⁺R₃Z⁻

wherein R₁ contains an alkyl, alkenyl or hydroxyalkyl group with 8 to 18carbon atoms, from 0 to 10 ethylene-oxy groups or from 0 to 2 glycerylunits; Y is a nitrogen, sulfur or phosphorous atom; R₂ is an alkyl orhydroxyalkyl group with 1 to 3 carbon atoms; x is 1 when Y is a sulfuratom and 2 when Y is a nitrogen or phosphorous atom; R₃ is an alkyl orhydroxyalkyl group with 1 to 5 carbon atoms and Z is a radical selectedfrom the group consisting of sulfate, sulfonate, carboxylate, phosphateor phosphonate.

Preferred amphoteric surfactants are amine oxides, for example cocodimethyl amine oxide. Preferred zwitterionic surfactants are betaines,and especially amidobetaines. Preferred betaines are C₈ to C₁₈ alkylamidoalkyl betaines, for example coco amido betaine. These may beincluded as co-surfactants, preferably present in an amount of from 0 to10 wt %, more preferably 1 to 5 wt %, based on the weight of the totalcomposition.

Preferred amphoteric or zwitterionic surfactants for incorporation inthe composition according to the present invention are betainesurfactants. Examples of these are mentioned in the following list.

The sulfatobetaines, such as 3-(dodecyldimethylammonium)-1-propanesulfate; and 2-(cocodimethylammonium)-1-ethane sulfate.

The sulfobetaines, such as:3-(dodecyldimethyl-ammonium)-2-hydroxy-1-propane sulfonate;3-(tetradecyl-dimethylammonium)-1-propane sulfonate; 3-(C₁₂-C₁₄alkyl-amidopropyldimethylammonium)-2-hydroxy-1-propane sulfonate; and3-(cocodimethylammonium)-1-propane sulfonate.

The carboxybetaines, such as (dodecyldimethylammonium) acetate (alsoknown as lauryl betaine); (tetradecyldimethylammonium) acetate (alsoknown as myristyl betaine); (cocodimethylammonium) acetate (also knownas coconut betaine); (oleyldimethylammonium) acetate (also known asoleyl betaine); (dodecyloxymethyldimethylammonium) acetate; and(cocoamido-propyldimethylammonium) acetate (also known ascocoamido-propyl betaine or CAPB).

The sulfoniumbetaines, such as: (dodecyldimethylsulfonium) acetate; and3-(cocodimethyl-sulfonium)-1-propane sulfonate.

The phosphoniumbetaines, such as 4-(trimethylphosphonium)-1-hexadecanesulfonate; 3-(dodecyldimethylphosphonium)-1-propanesulfonate; and2-(dodecyldimethylphosphonium)-1-ethane sulfate.

The compositions according to the present invention preferably comprisecarboxybetaines or sulphobetaines as amphoteric or zwitterionicsurfactants, or mixtures thereof. Especially preferred is laurylbetaine.

The treatment composition may comprise other ingredients commonly foundin detergent liquids. Especially polyester substantive soil releasepolymers, hydrotropes, opacifiers, colorants, perfumes, other enzymes,other surfactants, microcapsules of ingredients such as perfume or careadditives, softeners, polymers for anti redeposition of soil, bleach,bleach activators and bleach catalysts, antioxidants, pH control agentsand buffers, thickeners, external structurants for rheologymodification, visual cues, either with or without functional ingredientsembedded therein and other ingredients known to those skilled in theart.

Compositions

The composition is a laundry composition. Accordingly, suitably itcomprises one or more surfactants and/or optionally other ingredients.

Such compositions of the invention may be in dry solid form e.g.powdered, granules or tableted powders or liquid or gel form. It mayalso be in the form of a solid detergent bar. The composition may be aconcentrate to be diluted, rehydrated and/or dissolved in a solvent,including water, before use. The composition may also be a ready-to-use(in-use) composition.

In some cases, the composition is a liquid formulation.

The present invention is suitable for use in industrial or domesticfabric wash compositions, fabric conditioning compositions andcompositions for both washing and conditioning fabrics (so-calledthrough the wash conditioner compositions). The present invention canalso be applied to industrial or domestic non-detergent based fabriccare compositions, for example direct application e.g. roll-on orspray-on compositions which may be used as a pre-treatment of e.g.localised portions of fabric prior to a ‘main’ wash.

The enzymes may be present at 0-5 wt %, preferably 2-4 wt %, and mostpreferably 2.5-3.5 wt % of the composition (where wt % means percentageof the total weight of the composition).

The total protein concentration (of the total range of enzymes accordingto the invention) in the wash liquor may be from 0.01 to 10.0 mg/L, forexample from 2 to 5 mg/L.

Biosurfactant

Preferably, the composition includes a biosurfactant. The biosurfactantpreferably comprises a microbially-derived biosurfactant. Preferably itcomprises a glycolipid biosurfactant moiety which may be a rhamnolipidor sophorolipid or trehalolipid or a mannosylerythritol lipid (MEL) orcombinations thereof.

Alternatively or additionally the biosurfactant may comprise any shearthinning biosurfactant and in this respect, may extend to include anyshear thinning glycolipid biosurfactant mentioned above or any shearthinning cellobiose, peptide based biosurfactant, lipoprotein,lipopeptide e.g. surfactin, fatty acids e.g. corynomucolic acids(preferably with hydrocarbon chain C12-C₁₄), phospholipid (e.g.phosphatidylethanolamine produced by Rhodococcus erythropolis grown onn-alkane resulted in the lowering of interfacial tension between waterand hexadecane to less than 1 mN m⁻¹ and CMC of 30 mg L⁻¹ (Kretschner etal., 1982)), spiculisporic acid, polymeric biosurfactants includingemulsan, liposan, mannoprotein or polysaccharide-protein complexes orcombinations thereof.

The biosurfactant moiety may comprise one or more saccharide moietiessuch as sugar rings.

In some cases the biosurfactant is a mannosylerythritol lipid (MEL):

MEL-A: R₁=R₂=Ac; MEL-B: R₁=Ac, R₂=H; MEL-C: R₁=H; R₂=Ac: n=6-10.

In some cases, the biosurfactant is MEL-B.

In some cases, the biosurfactant moiety comprises a rhamnolipid.

In the case of rhamnolipids the rhamnolipid may comprise one or twosugar ring: mono-rhamnolipids having a single rhamnose sugar ring anddi-rhamnolipids, having two rhamnose sugar rings.

In the case of rhamnolipids, throughout this patent specification, theprefixes mono- and di-are used to indicate respectively to indicatemono-rhamnolipids (having a single rhamnose sugar ring) anddi-rhamnolipids (having two rhamnose sugar rings) respectively. Ifabbreviations are used R1 is mono-rhamnolipid and R2 is di-rhamnolipid.

The ratio of enzyme to biosurfactant surfactant may, for example, befrom 1:0.5 to 1:20, preferably from 1:0.5 to 1:10, such as from 1:0.5 to1:5.

The biosurfactant can be used to replace at least 50 wt. % of a totalsurfactant in the composition. In some cases, the stated biosurfactantis the only biosurfactant present. Preferably the biosurfactant ispresent at a level of 20-90 wt. % of the total surfactant and morepreferably the biosurfactant is present at 50-80 wt. % of the totalsurfactant and more preferably 50-75% wt. % of the total surfactant.

Other Ingredients

The enzymes may be the sole fabric treatment agent or other stainremoval agents may be incorporated.

Other detergent ingredients may be included including surfactants,builders, sequestering agents, hydrotropes, preservatives, complexingagents, polymers, stabilizers, perfumes, optical brighteners, or otheringredients such as e.g. fabric conditioners including clays, foamboosters, suds suppressors (anti-foams), anti-corrosion agents,soil-suspending agents, anti-soil redeposition agents, anti-microbials,tarnish inhibitors, or combinations of one or more thereof, providedthat these ingredients are compatible with the enzymes.

The fabric wash compositions may comprise a fabric wash detergentmaterial selected from biosurfactants, non-soap anionic surfactant,nonionic surfactants, soap, amphoteric surfactants, zwitterionicsurfactants and mixtures thereof.

It will be appreciated that the composition may include bothbiosurfactants and non-biosurfactants (in other words, a fabric washdetergent material, termed for ease a non-biosurfactant, and abiosurfactant. The composition may include a biosurfactant but not anon-biosurfactant. The composition may include a non-biosurfacant butnot a biosurfactant.

Any enzyme present in a composition may be stabilized using conventionalstabilizing agents, e.g., a polyol such as propylene glycol or glycerol,a sugar or sugar alcohol, lactic acid, boric acid, or a boric acidderivative, e.g., an aromatic borate ester, or a phenyl boronic acidderivative such as 4-formylphenyl boronic acid.

The following examples are provided by way of illustration and not byway of limitation.

Experimental (I) Psychromonas Ingrahami Lipase Cloning & ExpressionIncluding Sequence Information

A putative class 3 lipase from P. ingrahamii gene (PinLip) wasidentified used BLASTp. The PinLip protein sequence was searched againststructural characterised proteins in the PDB and showed highest homologyto Lipex from Rhizomucor miehei (Mucor miehei PDB: 5TGL) and Lipex(1TIB) with sequence identify of 14% to both. Protein sequence alignmentof PinLip to Lipex allowed the identification of Ser and Asp (FIG.1—serine and aspartate are conserved and identified in the sequencealignment, shown by underline) in the catalytic triad but the thirdresidue in the triad could not be identified because of very lowsequence identity in the C-terminal region of the lipases. Sequencealignments also allowed the identification of the PinLip lipase lidregion has also been identified (FIG. 1).

The gene coding for the PinLip into the pLATE 31 vector and successfullyexpressed in E. coli BL21 cells. Different expression conditions havebeen tested. An exemplary protocol is as follows: 1L LB mediasupplemented with 100 μgml⁻¹ ampicillin was inoculated and incubated for4 h at 37° C. with shaking at 180 rpm. When the OD reached 0.6 proteinproduction was induced by the addiction of 1.0 mM IPTG. Cultures werefurther incubated at 22° C. for 24 h with shaking at 180 rpm. After thistime the cells were harvested by centrifugation at 5000 g for 30 minutesat 4° C. The cell pellet was resuspended in 50 mM Tris-HCl pH 9.5 with 1mM CaCl₂ and the cells were harvested by centrifugation at 5000 g for 30minutes at 4° C. The cell pellet was stored at −20° C. until use.

Fermentation (Harvest) & Purification

The harvested cells were resuspended in 50 mM Tris-HCl pH 9.5 with 1 mMCaCl₂ and 1 M NaCl (Buffer A) using 5 ml/g of cells. [The selection ofpH at 9.5 was based on the fact that the theoretical isoelectric point(pl) of the PinLip (pl=7.21) was below this pH value and therefore theprotein would have a net negative charge at this pH value.] Lysozyme wasadded to the cells suspension at a final concentration of 0.1 mg/mL andthe suspension was stirred for 1 h at 4° C. The suspension was thensonicated using ten bursts of 20 seconds each with one minute rest onice between bursts. The lysate was then centrifuged at 24000 rpm for 1 hat 4° C. and the supernatant collected.

Purification by Hydrophobic Interaction (HIC)

The supernatant was loaded on Butyl FF column (size 1 mL). The columnwas previously equilibrated with 5 volume of Buffer A and the proteinwas eluted with a linear gradient between Buffer A and 50 mM Tris-HCl pH9.5 with 1 mM CaCl₂ (Buffer B). All eluted fractions were run on aSDS-PAGE gel. The fractions showing highest content of PinLip werepooled and concentrated. The protein was loaded on a DEAE FF column(size 1 mL) previously equilibrated with 5 volume of Buffer B and theprotein was eluted with a linear gradient between Buffer B and Buffer A.The fractions containing the pure PinLip were pooled and assayed forlipase activity.

As an alternative to the two-step purification procedure of HIC followedby DEAE, a one-step procedure based on a two-column tandem affinitysetting (maltose binding protein plus immobilized metal ion affinitychromatography for His-tagged PinLip) has been successfully applied. Thepurity of the final PinLip sample may be higher compared to the twocolumn purified product (data not shown).

Bioanalytics

SDS-PAGE was performed using a Bio-Rad Protean apparatus. The proteinsamples were initially boiled for 10 min at 100° C. after being dilutedwith Laemli loading buffer in order to denature the protein. Sampleswere then cooled and loaded on 10% acrylamide gel. Separation wasperformed in a running buffer following the running conditions: 120V,400 mA, for 90 min at room temperature. Protein bands on SDS-PAGE gelswere revealed by staining in “Comassie blue” prepared following themanufacturer's protocol. De-staining was performed at room temperaturefor 30 minutes to remove the excess dye. Results are shown in FIG. 2.The estimated purification yield is 5 mg/L.

Lipase Assay

For activity evaluation an assay based on the pNp release was performed(FIG. 3). Lipase was assayed in quadruplicate in a 96-well microtiterplate using p-nitrophenyllaurate (Sigma-Aldrich) as the substrate. 20 μLof PinLip (final concentration in well 25 ng/ml) was mixed with 100 μLof 50 mM Tris-HCl pH 8.5 containing 1 mM CaCl₂, and 0.01% of AOS plus 60μL of water and 20 μL of 1 mM pNp laurate. After 15 min of incubation atdifferent temperature, the OD 410 was measured against an enzyme-freecontrol. The values obtained have been calculated as μM of pNp releasedusing the standard curve shown in FIG. 4.

Results

The results of the lipase assay are shown in FIG. 5. FIG. 5 shows thatthe PinLip is active towards a wide range of pNp-ester coveringdifferent chain lengths and also that the increasing of temperature leadto an improved activity towards short/medium length esters.

End-Point Stain Removal Assays

The following soiled cloth samples were hole-punched into discs andtransferred to 300 μl 96 well plates:

Lipase sensitive stains: CS61—Beef fat stain and C646B—Used fry fat (CFTBV)

Laundry Enzymes (Novozymes):

-   -   Lipex 100 L (Novozymes)    -   T. lanuginosa lipase (Sigma-Aldrich)

Enzyme

-   -   Psychromonas ingrahamii Lipase (PinLip)

Procedure:

Test Mixture: Total wash volume=200 μlSoluble enzyme PinLip/Lipex (20 mg/L in assay well)=20 μlBlackbull formulation 8 g/L stock (0.8 g/L final in assay well)=20 μl

Prenton Water=160 μl

In both cases the enzyme was added last. Two sets of reactions wereincubated at 20 and 40 degrees respectively for 1 hour with shaking at250 rpm.

The assay plates were dried overnight.

After drying, the stain plates were digitally scanned and their deltaEmeasured. This value is used to express cleaning effect and is definedas the colour difference between a white cloth and that of the stainedcloth after being washed. Mathematically, the definition of deltaE is:

deltaE=[(Δ_(L))²+(Δ_(a))²+(Δ_(b))²]^(1/2)

wherein Δ_(L) is a measure of the difference in darkness between thewashed and white cloth; Δ_(a) and Δ_(b) are measures for the differencein redness and yellowness respectively between both cloths. From thisequation, it is clear that the lower the value of deltaE, the whiter thecloth will be. With regard to this colour measurement technique,reference is made to Commission International de l'Eclairage (CIE);Recommendation on Uniform Colour Spaces, colour difference equations,psychometric colour terms, supplement no. 2 to CIE Publication, no. 15,Colormetry, Bureau Central de la CIE, Paris 1978.

Results

In FIG. 6 the cleaning effect is expressed in the form of a stainremoval index (SRI): SRI=100−deltaE. The higher the SRI the cleaner thecloth, SRI=100 (white). PinLip shows a better cleaning effect comparedto Lipex in high (˜30%) and low total surfactancy (˜15%).

Dose Dependant Wash Study

As further investigation the same end point wash studies has beenconducted varying the concentration of enzyme in order to find the bestdosage (FIG. 7). These results show that PinLip is superior in cleaningcompared to benchmark Lipex at different enzyme doses used, especiallyin the range between 20 and 2.5 mg/L.

Impact of Rhamnolipids on Enzymatic Cleaning (No Formulation)

In order to understand the effect of rhamnolipids on cleaning effect ofenzymes, different rhamnolipids i.e. R1 (mono rhamnolipid), 4R2 (dirhamnolipid) and MEL-B (Mannnosylerythritol lipid B), 1614 Mel(multi-MEL components) were screened at different concentration levelsin presence and absence of enzymes in wash study. A stock concentrationof 70% (active) rhamnolipid was prepared for wash study and dilutedusing mili-Q water to achieve a working stock concentration in range of20% to 1.25%. Wash study was carried out in similar way as mentioned inabove sections.

Test Mixture: Total wash volume=200 μlSoluble enzyme PinLip/Lipex (20 mg/L in assay well=20 μlRhamnolipid 8 g/L stock (0.8 g/L final in assay well)=20 μl

Prenton Water=160 μl

In both cases the enzyme was added last. Assay plates were incubated at20 degrees for 1 hour with shaking at 250 rpm.

4R2 RL increases its cleaning performance on its own with increasingconcentration, so that the contribution from the enzymes is minimalabove 0.05% 4R2 RL. In 4R2 RL, PinLip and Lipex showed equivalent goodcleaning performance in the presence of R2, even better at low RLconcentration.

In R1 RL, PinLip shows better synergistic cleaning performance (enzymeplus RL) than the benchmark enzyme, Lipex. See FIG. 8 a.

In presence of 1614 MEL and MEL-B in combination with PinLip showed overthe full dosage range superior cleaning compared to the MEL Lipexcombination. See FIGS. 8b and 8 c.

Partial Detergency Substitution in Formulation by RL

In order to study the effect of rhamnolipid in presence of LAS/SLES/NIon cleaning effect of lipases, a range of formulations were prepared byadding rhamnolipids at different concentration level in presence of LAS,SLES, and NI. The ratio of LAS, SLES, and NI was 2:1:3 in the preparedformulations. The wash study was carried out as mentioned in abovesection.

TABLE 1 Formulation containing different level of biosurfactant(rhamnolipids) and chemical surfactant to give 100% active surfactantsystem. In given chemical surfactant system the ratio of LAS, SLES andNI was 2:1:3. Surfactant Biosurfactant (%) (LAS:SLES:NI)(2:1:3)(%) Total10 90 100 20 80 100 30 70 100 40 60 100 50 50 100

TABLE 2 Amounts of various components in the biosurfactant andsurfactant blend. Biosurfactant (g) LAS (g) SLES (g) NI (g) Total(g) in1000 ml 0.8 2.4 1.2 3.60 8 1.6 2.13 1.07 3.20 8 2.4 1.87 0.93 2.80 8 3.21.60 0.80 2.40 8 4 1.33 0.67 2.00 8

Rhamnolipids (R1—one rhamnose molecule, 4R2—two rhamnose moleculescontaining), and lipases were tested at a c dose in end point removalassay using CS61 Beef fat stain. 8 replicates were performed in parallelon the same 96 well plate. The experiment was carried out at 20 degrees.The plates were scanned and the SRI values calculated. The results areshown in FIG. 9a as a bar chart displaying the average SRI forreplicates. Error bars display standard deviation between the replicatesfor each condition.

In a R1 modified chemical surfactant blend, PinLip shows better cleaningthan Lipex up to 30% R1 content in the formulation (strong positiveimpact at low concentration in formulation).

In a R2 modified chemical surfactant blend, the results revealed thatPinLip shows better cleaning than Lipex below 20% R2 content, at highercontent of R2, Lipex and PinLip showed parity in cleaning and above 30%not better any more than the modified formulation. The results are shownin FIG. 9b as a bar chart displaying the average SRI for replicates.Once again, error bars display standard deviation between the replicatesfor each condition.

1. A lipase having a sequence identity of at least 70% with SEQ. ID. 2.2. A laundry composition comprising a lipase having a sequence identityof at least 70% with SEQ. ID.
 2. 3. A laundry composition comprising alipase from Pyschromonas ingrahamii.
 4. The laundry composition of claim3, wherein the lipase from Pyschromonas ingrahamii is a putative class 3lipase.
 5. The composition of claim 1, wherein the composition furthercomprises a biosurfactant, optionally wherein the biosurfactant is aglycoplipid.
 6. The laundry composition of claim 5, wherein thebiosurfactant is selected from a rhamnolipid, sophorolipid,trehalolipid, and a mannosylerythritol lipid (MEL), and combinationsthereof.
 7. The laundry composition of claim 5, wherein thebiosurfactant is a rhamnolipid, optionally wherein the rhamnolipidcomprises at least 50 wt. % monorhamnolipid, optionally wherein therhamnolipid comprises at least 80 wt. % monorhamnolipid.
 8. The laundrycomposition of claim 5, wherein the biosurfactant is amannosylerythritol lipid, optionally wherein the mannosylerythritollipid comprises at least 50 wt. % mannosylerythritol lipid B, optionallywherein the mannosylerythritol lipid comprises at least 80 wt. %mannosylerythritol lipid B.
 9. The laundry composition of claim 1,wherein the total surfactant content of the composition is 30 wt. % orless.
 10. The laundry composition of claim 1, wherein the compositioncomprises one or more ingredients selected from builders, sequesteringagents, hydrotropes, preservatives, complexing agents, polymers,stabilizers, perfumes, optical brighteners, or other ingredients such ase.g. fabric conditioners including clays, foam boosters, sudssuppressors (anti-foams), anti-corrosion agents, soil-suspending agents,anti-soil redeposition agents, anti-microbials, and tarnish inhibitors,and combinations of one or more thereof.
 11. A method of launderingarticles, the method comprising washing articles in an aqueous washliquor containing a laundry composition according to claim 1, whereinthe temperature of the wash liquor is 25° C. or less.
 12. The method ofclaim 11, wherein the temperature of the wash liquor is 15-25° C. 13.The method of claim 11, wherein the concentration of psychrophiliclipase in the wash liquor is 2.5 to 20 mg/L.
 14. The method of claim 11,wherein the concentration of biosurfactant in the wash liquor is 0.001to 1 wt. %, optionally wherein the concentration of biosurfactant in thewash liquor is 0.01 to 0.2 wt. %